This project was initiated to study the molecular genetics of cell cycle associated genes in C. neoformans. In 2006-2007, we deleted the TUP1 gene, a fungal global repressor known to regulate the rate of cell proliferation, mating and morphogenesis, in H99, a serotype A strain of C. neoformans. During the period of 2008-2009, we found other pleiotropic phenotypes in serotype A tup1 null strains that had not been observed in serotype D strains. These include increased susceptibility to azole drugs, the most widely used antimycotics to treat cryptococcosis, sensitivity to SDS and a reduction in the formation of melanin, one of the major virulence factors in C. neoformans. These results suggested the pathobiological importance of TUP1 in C.neoformans.C. neoformans is notorious for not behaving like other yeasts in many aspect. One of such pattern is found in cell cycle and a method for synchronization has not been achieved. In many studies , cell cycle synchronization is required to resolve the questions on biological phenomenon that is associated with cell cycle. During 2009-2010, we were able to use a sucrose gradient to isolate cells enriched in G1 phase (>95% purity), but these cells did not exit the G1 phase in synchronized fashion. We used chemicals that have previously been used to achieve synchronization in other organisms, such as hydoxyurea, nocodazole, benomyl, azetidine 2-carboxylic acid and methylmethane sulfonate, but without success. Mutants of cell cycle genes known to cause cell cycle arrest in other systems were also generated in our laboratory. We have constructed a temperature sensitive allele of CDC28 and an ATP-analog sensitive allele of CDC15. However, both mutants failed to cause specific arrest in the cell cycle at restricted conditions. We are currently focusing on pheromone-mediated cell cycle arrest and are testing the G1 exit pattern in differennt strains in order to achieve synchronization of the cell cycle. We expanded our research further during 2010-2011 to uncover the signaling pathways that are pertinent to pathogenicity and drug resistance in C. neroformans. The first study focused on the signaling pathways involved in azole resistance by screening a deletion library of the serotype A genome sequenced strain, H99. Several genes involved in the conserved PDK1 and PKC signaling pathways were found to contribute to the basal tolerance of FLC. Moreover, genes in these two pathways were also found to play different roles in response to salt and rapamycin treatment as well as in controlling homeostasis of complex sphingolipids. We also found evidence that suggests involvement of the TOR pathway in the basal tolerance of FLC. Deletions of PDK1, SIN1, or YPK1 but not MPK1 were deleterious to cell viability in the presence of compounds that inhibit sphingolipid biosynthesis. These mutants accumulated higher amounts of FLC compared with wild-type suggesting a malfunction of the influx/efflux systems in the mutants. We speculate that the FLC hypersensitivity phenotype of pdk1, sin1, and ypk1 is associated with impaired membrane composition due to modified sphingolipid content. Interestingly, the reduced virulence of these three strains in mice suggests that cryptococcal PDK1, PKC, and likely the TOR pathways not only play important roles in managing stress exerted by FLC but also by the host's environment. During 2011-2012, we discovered that Sac6, an actin-binding protein, plays an important role in C. neoformans cell growth at low oxygen conditions. During 2013-2014,we focused on the signalling pathway involved in oxygen sensing by C. neoformans and found that Ras1, Cdc24 and Ptp3 pathways have an active role in the fungal growth under low oxygen stress. During 2014-2016, we analyzed the phospho-proteomics from the mutants of ptp3, hog1 and ptp3/hog1. To broaden our studies on the molecular analysis of signal pathway, we expanded our analyses to the regulation of efflux pump genes and D-amino acid utilization in 2016. First, we deciphered the roles of three ABC transporters, Afr1, Afr2 and Mdr1 in C. neoformans H99 and C. gattii R265. We found that fluconazole treatment caused greater than two-fold increase in the transcription levels for both CnAFR1 and CnAFR2, but not for CnMDR1 in H99. In contrast, all three efflux pump genes in R265 showed significantly increased transcription levels in response to fluconazole treatment. To identify the transcription factors regulating the expression of the efflux pump genes, we screened the H99 transcriptional factor deletion library and isolated several mutants manifesting altered fluconazole sensitivity or increase in the frequency of fluconazole heteroresistance. Gene expression studies suggested that the expression of AFR1, AFR2, MDR1 and ERG11 is independently regulated by different transcription factors. Second, we previously found that myo5 mutant failed to grow on D-proline as a sole nitrogen source and resulted in increased expression of the major gene involved in utilization of many D-amino acids, RDAO2, in C. gattii. In 2017, we found accumulation of D-proline or D-alanine increased significantly in myo5 deletant which may explain the growth defect of myo5 mutant on D-proline and D-alanine. We further demonstrated that myo5 mutant had increased membrane permeability towards many substrates including several antifungal agents in C. gattii. Furthermore, the involvement of Myo5 in membrane permeability is conserved among C. neoformans, C. gattii, Candida albicans and Schizosaccharomyces pombe.